Temperature-Dependent Development of Pasteuria penetrans in Meloidogyne arenaria

Pasteuria penetrans is a promising biological control agent of plant-parasitic nematodes. This study was conducted to determine effects of temperature on the bacterium''s development in Meloidogyne arenaria. Developmental stages of P. penetrans were viewed with a compound microscope and verified with scanning electron microscopy within each nematode at 100 accumulated degree-day intervals by tracking accumulated degree-days at three temperatures (21, 28, and 35 °C). Five predominant developmental stages of P. penetrans were identified with light microscopy: endospore germination, vegetative growth, differentiation, sporulation, and maturation. Mature endospores were detected at 28, 35, and >90 calendar days at 35, 28, and 21 °C, respectively. The number of accumulated degree-days required for P. penetrans to reach a specific developmental stage was different for each temperature. Differences were observed in the development of P. penetrans at 21, 28, and 35 °C based on regression values fitted for data from 100 to 600 accumulated degree-days. A linear response was observed between 100 to 600 accumulated degree-days; however, after 600 accumulated degree-days the rate of development of P. penetrans leveled off at 21 and 28 °C, whereas at 35 °C the rate decreased. Results suggest that accumulated degree-days may be useful only in predicting early-developmental stages of P. penetrans.     

Biological Control of Meloidogyne incognita by Paecilomyces lilacinus and Pasteuria penetrans

The root-knot nematode Meloidogyne incognita was controlled more effectively and yields of host plants were greater when Paecilomyces lilacinus and Pasteuria penetrans were applied together in field microplots than when either was applied alone. Yields of winter vetch from microplots inoculated with the nematode and with both organisms were not statistically different from yields from uninoculated control plots.     

Suitability of Zucchini and Cucumber Genotypes to Populations of Meloidogyne arenaria,M. incognita,and M. javanica

The host suitability of five zucchini and three cucumber genotypes to Meloidogyne incognita (MiPM26) and M. javanica (Mj05) was determined in pot experiments in a greenhouse. The number of egg masses (EM) did not differ among the genotypes of zucchini or cucumber, but the eggs/plant and reproduction factor (Rf) did slightly. M. incognita MiPM26 showed lower EM, eggs/plant, and Rf than M. javanica Mj05. Examination of the zucchini galls for nematode postinfection development revealed unsuitable conditions for M. incognita MiPM26 as only 22% of the females produced EM compared to 95% of the M. javanica females. As far as cucumber was concerned, 86% of the M. incognita and 99% of the M. javanica females produced EM, respectively. In a second type of experiments, several populations of M. arenaria, M. incognita, and M. javanica were tested on zucchini cv. Amalthee and cucumber cv. Dasher II to assess the parasitic variation among species and populations of Meloidogyne. A greater parasitic variation was observed in zucchini than cucumber. Zucchini responded as a poor host for M. incognita MiPM26, MiAL09, and MiAL48, but as a good host for MiAL10 and MiAL15. Intraspecific variation was not observed among the M. javanica or M. arenaria populations. Cucumber was a good host for all the tested populations. Overall, both cucurbits were suitable hosts for Meloidogyne but zucchini was a poorer host than the cucumber.     

Suppression of Meloidogyne incognita and M. javanica by Pasteuria penetrans in Field Soil

The role of Pasteuria penetrans in suppressing numbers of root-knot nematodes was investigated in a 7-year monocuhure of tobacco in a field naturally infested with a mixed population of Meloidogyne incognita race 1 and M. javanica. The suppressiveness of the soil was tested using four treatments: autoclaving (AC), microwaving (MW), air drying (DR), and untreated. The treated soil bioassays consisted of tobacco cv. Northrup King 326 (resistant to M. incognita but susceptible to M. javanica) and cv. Coker 371 Gold (susceptible to M. incognita and M. javanica) in pots inoculated with 0 or 2,000 second-stage juveniles of M. incognita race 1. Endospores of P. penetrans were killed by AC but were only slightly affected by MW, whereas most fungal propagules were destroyed or inhibited in both treatments. Root galls, egg masses, and numbers of eggs were fewer on Coker 371 Gold in MW, DR, and untreated soil than in AC-treated soil. There were fewer egg masses than root galls on both tobacco cultivars in MW, DR, and untreated soil than in the AC treatment. Because both Meloidogyne spp. were suppressed in MW soil (with few fungi present) as well as in DR and untreated soil, the reduction in root galling, as well as numbers of egg masses and eggs appeared to have resulted from infection of both nematode species by P. penetrans.     

Suppression of Meloidogyne arenaria Race 1 by Soil Application of Endospores of Pasteuria penetrans

The potential of Pasteuria penetrans for suppressing Meloidogyne arenaria race 1 on peanut (Arachis hypogaea) was tested over a 2-year period in a field microplot experiment. Endospores of P. penetrans were mass-produced on M. arenaria race 1 infecting tomato plants. Endospores were inoculated in the first year only at rates of 0, 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively, into the top 20 cm of microplots that were previously infested with M. arenaria race 1. One peanut seedling was planted in each microplot. In the first year, root gall indices and pod galls per microplot were significantly reduced by 60% and 95% for 100,000 endospores/g of soil, and 20% and 65% for 10,000 endospores/g of soil, respectively. Final densities of second-stage juveniles (J2) in soil were not significantly different among the treatments. The number of endospores attached to J2 and percentage of J2 with attached endospores significantly increased with increasing endospore inoculation levels. Pasteuria penetrans significantly reduced the densities of J2 that overwintered. In the second year, root and pod gall indices, respectively, were significantly reduced by 81% and 90% for 100,000 endospores/g of soil, and by 61% and 82% of 10,000 endospores/g of soil. Pod yields were significantly increased by 94% for 100,000 and by 57% for 10,000 endospores/g of soil, respectively. The effect of P. penetrans on final densities of J2 in soil was not significant. Regression analyses verified the role of P. penetrans in the suppression of M. arenaria. The minimum number of endospores required for significantly suppressing M. arenaria race 1 on peanut was 10,000 endospores/g of soil.     

Effects of Fluctuating Temperatures and Different Host Plants on Development of Pasteuria penetrans in Meloidogyne javanica

Greenhouse and growth room experiments were conducted to investigate the effect of host plant in relation to different nematode inoculum levels, and temperature fluctuations on the development of Pasteuria penetrans. Host plant affected the development of P. penetrans indirectly through its effect on nematode development. Endospores collected from Meloidogyne javanica females reared on different hosts did not show any differences in subsequent attachment and infectivity. The numbers of endospores produced per infected female were reduced with increasing numbers of females parasitizing okra and tomato roots. Fluctuating temperatures retarded the development of P. penetrans. The life cycle of the parasite was completed faster at approximately constant temperatures close to 30 °C than when the temperature fluctuated away from 30 °C. The temperature of irrigation water did not affect the duration of life cycle of P. penetrans.     

Effect of Temperature on Attachment,Development, and Interactions of Pasteuria penetrans on Meloidogyne incognita

The effect of temperature (10, 20, 25, 30, and 35 C) on attachment and development of Pasteuria penetrans on Meloidogyne arenaria race 1 was elevated in growth chambers. The greatest attachment rate of endospores of P. penetrans occurred on second-stage juveniles at 30 C. The bacterium developed more quickly within its host at 30 and 35 C than at 25 C or below. The development of the bacterium within the nematode female was divided into nine recognizable life stages, which ranged from early vegetative thalli to mature sporangia. Mature sporangium was the predominant life stage observed after 35, 40, 81, and 116 days at 35, 30, 25, and 20 C, respectively. The body width and length of M. arenaria females infected with P. penetrans were smaller initially than the same dimensions in uninfected females, but became considerably larger over time at 25, 30, and 35 C. This isolate of P. penetrans also parasitized and completed its life cycle in males of M. arenaria.     

Estimating Incidence of Attachment of Pasteuria penetrans Endospores to Meloidogyne spp. with Tally Thresholds

Pasteuria penetrans has .been identified as an important biological control agent of root-knot nematodes. In this study the use of tally thresholds was evaluated for estimating P. penetrans endospore attachment to second-stage juveniles (J2) of Meloidogyne spp. A tally threshold (T) is defined as the maximum number of individuals in a sample unit that may be treated as absent based on binomial sampling. Three different data sets that originated from centrifugal bioassay, incubation bioassay, and field experiments were investigated. The data sets each contained 70, 33, and 111 estimates of the mean number of endospores attached per J2 (m), respectively. Empirical relationships between m and proportions of J2 with ≤T endospores attached (P_T) were developed using parameters from the linear regression of ln(m) on P_T (0 < P_T < 1): ln(m) = a + b P_T, T was set to 0, 1, 2, 3, 4, 5, 8, and 10 endospores/J2. The results indicated that the variances of linear equations tended to decrease with increasing T values for all three data sets. T values of 0, 1, 8, and 10 endospores/J2 for centrifugal bioassay and incubation bioassay, and of 0, 1, 2, and 3 endospores/J2 for field experiments were associated with an r² of >= 0.8. These T values were robust for estimating m from P_T, reducing the variability as well as the time and effort spent in estimating the mean number of endospores attached per J2.     

Quantification of Endospore Concentrations of Pasteuria penetrans in Tomato Root Material

Six methods for quantification of the endospore concentrations of Pasteuria penetrans from tomato roots are described. Mortar disruption and machine disruption methods gave the highest estimations (endospores per gram of root material) of 83.7 and 79.0 million, respectively. These methods were significantly superior to incubation bioassay (47.7 million), enzymatic disruption (32.1 million), and enzymatic disruption + flotation (25.8 million) methods. A centrifugation bioassay method gave the lowest estimation of 12.7 million.     

Susceptibility of Several Common Subtropical Weeds to Meloidogyne arenaria,M. incognita,and M. javanica

Experiments were conducted in the greenhouse to assess root galling and egg production of three root-knot nematode species, Meloidogyne arenaria, M. incognita, and M. javanica, on several weeds common to Florida agricultural land. Weeds evaluated were Amaranthus retroflexus (redroot pigweed), Cyperus esculentus (yellow nutsedge), Eleusine indica (goosegrass), Portulaca oleracea (common purslane), and Solanum americanum (American black nightshade). Additionally, although it is recommended as a cover crop in southern regions of the U.S., Aeschynomene americana (American jointvetch) was evaluated as a weed following the detection of root galling in a heavy volunteer infestation of an experimental field in southeastern Florida. Weeds were propagated from seed and inoculated with 1000 nematode eggs when plants reached the two true-leaf stage. Tomato (Solanum lycopersicum ‘Rutgers’) was included as a positive control. Aeschynomene americana and P. oleracea roots supported the highest number of juveniles (J₂) and had the highest number of eggs/g of root for all three species of Meloidogyne tested. However, though P. oleracea supported very high root levels of the three nematode species tested, its fleshy roots did not exhibit severe gall symptoms. Low levels of apparent galling, combined with high egg production, increase the potential for P. oleracea to support populations of these three species of root-knot nematodes to a degree that may not be appropriately recognized. This research quantifies the impact of P. oleracea as a host for M. arenaria, M. incognita, and M. javanica compared to several other important weeds commonly found in Florida agricultural production, and the potential for A. americana to serve as an important weed host of the three species of root-knot nematode tested in southern regions of Florida.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.     

Population Dynamics of Meloidogyne arenaria and Pasteuria penetrans in a Long-Term Crop Rotation Study

The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities increased under intensive cropping of hosts for M. arenaria, but the GH population of the nematode was not receptive to spore attachment. However, previously, the GH population was very receptive to spore acquisition from this field site. One explanation for this inconsistency is that the M. arenaria population in the field became resistant to the dominant subpopulation of P. penetrans that had been present, and this led to the selection of a different subpopulation of the bacterium that is incompatible with the GH population.     

Distribution and Downward Movement of Pasteuria penetrans in Field Soil

Endospores of Pasteuria penetrans were evaluated for their vertical distribution in field soil and their downward movement through soil in the laboratory. In the field trial, the number of endospores attached to second-stage juveniles (J2) of Meloidogyne arenaria race 1 varied greatly in different soil depths. There were higher percentages of J2 with endospores attached in former weed fallow plots during the first 3 years of growing peanut than in former bahiagrass and rhizomal peanut plots (P ≤ 0.05). In weed fallow plots a higher average number of endospores per J2 were maintained in all depths, upper three depths, and upper four depths in 1999, 2000, and 2001, respectively (P ≤ 0.05). However, in 2002, there were no differences in the percentages of J2 with endospores attached and in the average of the numbers of endospores per J2 among the treatments (P > 0.05). In laboratory trials, P. penetrans endospores were observed to move throughout the soil through the percolation of water. After one application of water, some endospores were detected 25 to 37.5 cm deep. Endospores were present at the greatest depth, 37.5 to 50 cm, after the third application of water. These results indicate that rain or water applications by irrigation are likely to move endospores to deeper levels of the soil, but the majority of endospores remain in the upper 0-to-30-cm depth.     

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and α-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.     

Interrelationships of Meloidogyne arenaria and M. incognita on Tolerant Soybean

Reproduction of Meloidogyne arenaria race 2 was excellent on Centennial, Govan, and Kirby soybeans, the latter two of which have tolerance to this species. The M. incognita race 1 isolate reproduced poorly on Centennial, especially at the higher of two temperature regimes. Numbers of galls and egg masses of M. arenaria plus M. incognita in simultaneous equivalent infestations on Centennial did not differ from sequential infestations in which M. arenaria was added first and M. incognita was added to the same pots, 1,2, or 3 weeks later. However, at both 25 and 30 C, suppression of galls and egg masses occurred when inoculation of M. incognita preceded that of M. arenaria by 2 weeks. Generally, M. arenaria reproduced well at 25 or 30 C, whereas M. incognita reproduced better at 30 C. Kirby was tolerant to either nematode species at 25 and 30 C, but in combined infestations of M. arenaria and M. incognita there was evidence of synergistic growth suppression. Govan was tolerant of M. arenaria at 25 C but not at 30 C. Moreover, general plant growth was less vigorous for Govan at the higher temperature, whereas Centennial was much more vigorous at this temperature. Kirby grew equally well at both temperatures.     

Impact of Paecilomyces lilacinus Inoculum Level and Application Time on Control of Meloidogyne incognita on Tomato

Microplot experiments were conducted to evaluate the effects of inoculum level and time of application of Paecilomyces lilacinus on the protection of tomato against MeIoidogyne incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus-infested wheat kernels per microplot which resulted in a threefold and fourfold increase in tomato yield, respectively, compared with tomato plants treated with this nematode alone. Greatest protection against this pathogen was attained when P. lilacinus was delivered into soil 10 days before planting and again at planting. Yield was increased twofold compared with yield in nematode-alone plots and plots with M. incognita plus the fungus. Percentages of P. lilacinus-infected egg masses were greatest in plots treated at midseason or at midseason plus an early application, compared with plots treated with the fungus 10 days before planting and (or) at planting time.     

Evaluation of Steam and Soil Solarization for Meloidogyne arenaria Control in Florida Floriculture Crops

Steam and soil solarization were investigated for control of the root-knot nematode Meloidogyne arenaria in 2 yr of field trials on a commercial flower farm in Florida. The objective was to determine if preplant steam treatments in combination with solarization, or solarization alone effectively controlled nematodes compared to methyl bromide (MeBr). Trials were conducted in a field with naturally occurring populations of M. arenaria. Treatments were solarization alone, steam treatment after solarization using standard 7.6-cm-diameter perforated plastic drain tile (steam 1), steam treatment following solarization using custom-drilled plastic drain tile with 1.6-mm holes spaced every 3.8 cm (steam 2), and MeBr applied at 392 kg/ha 80:20 MeBr:chloropicrin. Drain tiles were buried approximately 35 cm deep with four tiles per 1.8 by 30 m plot. Steam application followed a 4-wk solarization period concluding in mid-October. All steam was generated using a Sioux propane boiler system. Plots were steamed for sufficient time to reach the target temperature of 70°C for 20 min. Solarization plastic was retained on the plots during steaming and plots were covered with a single layer of carpet padding to provide additional insulation. The floriculture crops larkspur (Delphinium elatum and Delphinium × belladonna), snapdragon (Antirrhinum majus), and sunflower (Helianthus annuus) were produced according to standard commercial practices. One month after treatment in both years of the study, soil populations of M. arenaria were lower in both steam treatments and in MeBr compared to solarization alone. At the end of the season in both years, galling on larkspur, snapdragon, and sunflowers was lower in both steam treatments than in solarization. Both steam treatments also provided control of M. arenaria in soil at the end of the season comparable to, or exceeding that provided by MeBr. Both steam treatments also reduced M. arenaria in snapdragon roots comparable to, or exceeding control with MeBr. Meloidogyne arenaria in soil increased in solarization alone. Solarization alone also had higher gall ratings on larkspur, snapdragon, and sunflower than all other treatments. Steam provided excellent control of M. arenaria in this study.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.